Project Summary Ion channels are expressed in every cell in the body and are involved in critical and diverse physiological processes including fast neurotransmission, establishing and maintaining cardiac rhythms, and solute balance. The importance and diversity of ion channel function results both in ion channel-related pathologies but also in a number of ion channel-targeted therapies. Though ion channels have been the targets of intense research over the last three decades, much remains to be understood regarding the roles of specific ion channels in normal physiological processes and disease. A substantial reason for our lack of understanding of the roles of specific ion channels process is the lack of potent and selectively pharmacological tools targeting these channels. The G-protein Coupled Inward Rectifying Potassium K+ Channels (GIRK) are prime examples of an ion channel family that has been the focus of research for nearly two decades and are thought to be potential targets for numerous indications spanning from atrial fibrillation to analgesia. However, there is but one highly potent and selective GIRK inhibitor and no potent and selective GIRK activators. GIRKs are comprised of homo and heteromeric combinations of four subunits (GIRK 1-4). These subunits are expressed in numerous tissues in the CNS and the periphery. Very recently we have used thallium (Tl+) flux- based screening to discover the first potent and selectively activators of a GIRK channel comprised of GIRK1/2 subunits. Surprisingly, none of the ~ 100 GIRK 1/2 activators evaluated thus far show any activity at non-GIRK 1 containing GIRKs (GIRK 2, GIRK 2/3). Thus, we propose to perform a high-throughput screen to discover the first selective activators of the GIRK 2/3 subunit combination. The GIRK 2 and GIRK2/3 subunit combinations have a relatively restricted expression pattern in mid-brain structures such as the ventral tegmental area (VTA) and discrete subcellular localization compared to GIRK 1-containing GIRKs. Though there is building research that implicates these channels in reward and addiction-related circuitry, most of understanding of GIRKs role remains uncertain due to the lack of selective pharmacological tools. Building upon our success in discovering small molecule modulators of GIRK 1/2 using Tl+ flux-based high-throughput screening, we are proposing to screen a 160,000 sample compound collection and characterize hits using a well-developed set of confirmatory, mechanism of action, and selectivity screens designed to identify compounds with the capacity to be developed as subunit-selective GIRK probes. These probes will be immediately useful to begin understanding mechanisms of action and selectivity between GIRK 1 and non-GIRK1 containing GIRKs. These probes will also advance as candidates for in vivo probe development to enable the investigation of GIRK's roles in physiology and exploration of its therapeutic potential for a variety of important indications including addiction, pain, and epilepsy.